Humanised baculovirus

ABSTRACT

The invention relates to a genetically engineered baculovirus wherein said virus is engineered to target therapeutic agents to cells, typically cancer cells, for example prostate cancer cells.

The invention relates to a baculovirus wherein the baculovirus genomecomprises a nucleic acid molecule encoding a therapeutic agent and anucleic acid molecule which encodes a polypeptide wherein saidpolypeptide functions to target the baculovirus to at least one celltype; methods of treatment using said baculovirus and pharmaceuticalcompositions comprising said baculovirus.

Gene therapy involves the transfer, and optionally the stable insertion,of new genetic information into cells for the therapeutic treatment ofdisease. The main issues with respect to gene therapy relate to theefficient targeting of nucleic acid to cells and the establishment ofhigh level transgene expression in selected tissues. A number ofmethodologies have been developed which purport to facilitate either orboth of these requirements. For example, U.S. Pat. No. 6,043,339discloses the use of signal peptides which when fused to a nucleic acidcan facilitate the translocation of the linked nucleic acid across cellmembranes. U.S. Pat. No. 6,083,714 discloses a combined nucleic acid andtargeting means which uses the polycation poly-lysine coupled to anintegrin receptor thereby targeting cells expressing the integrin.EP1013770 discloses the use of nuclear localisation signals (NLS)coupled to oligonucleotides. The conjugate may be covalently linked tovector DNA and the complex used to transfect cells. The NLS sequenceserves to facilitate the passage of the vector DNA across the nuclearmembrane thereby targeting gene delivery to the nucleus.

A range of viral based vectors have been used to successfully transfectmammalian cell lines. These include adenovirus, adenovirus-associatedvirus, papovaviruses and vacciniavirus. These viral based vectors haveconsiderable disadvantages. Adenovirus vectors are well established ingene therapy trials, although recent difficulties in the USA mayrestrict their use. (Wickham T J, Gene therapy, 7: 110, 2000). The majorproblems appear to be non-selective cytotoxicity (particularly in theliver) and pre-existing immune responses against the virus. Thecytolytic T cell response induced against adenovirus capsid-derivedpeptides has been shown to mediate the destruction of vector transducedcells and has been associated with localised tissue damage andinflammation. (Gilgenkrantz, H. et al. (1995) Hum Gene Ther 6,1265-1274; Yang, Y. and Wilson, J. M. (1995) J Immunol 155, 2564-2570).The possibility of recombination with endogenous infecting adenovirus,particularly at high input dose, is also a potential safety concern.

Limitations to the amount of extra genetic material inserted intorecombinant viruses are imposed by the defined size of the adenoviruscapsid. Adenoviruses will recombine with pre-existing material; apotential drawback where endogenous adenovirus is wide spread in thehuman population. Similarly it has been demonstrated that adenovirusvectors have the ability to aid the replication of related endogenoushuman viruses.

Safety concerns are also associated with the clinical use of HerpesSimplex Virus. Lytic replication of the virus in the human brain hasbeen linked to encephalitis. (Latchman, D. S. (1994). Mol Biotechnol 2,179-195).

Although retroviral vectors are widely used in clinical trials, a numberof disadvantages are associated with these vectors. Integration intocells is random, a major safety concern. Use of these vectors islimited, as they require dividing cells for infectivity.

An alternative vector, which has been shown to infect mammalian cells,is the baculovirus. Baculovirus is a rod form virus and thereforelimitations to the amount of genetic material inserted into recombinantbaculovirus is not as limiting as those imposed by adenovirus capsid.

The baculovirus will not express its own genes from insect-specificpromoters in human cells. This is an attractive feature since thebaculovirus will not provoke an immune response as a consequence ofviral gene expression of virally encoded genes. However, insertion of amarker or therapeutic gene under control of a mammalian promoter allowshigh level expression of the transgene. Unlike the adenovirus vector,baculovirus will not recombine with pre-existing material. Infectionwith baculovirus will not facilitate the replication of endogenous humanviruses, as has been demonstrated with adenovirus vectors. In contrastto many of the other therapeutic viruses, baculoviruses can be grown ina serum free culture media in large quantities. This method ofproduction can be readily scaled up to industrial level and removes thepotential hazards of serum contamination of the therapeutic agent withviral and prion agents. Most importantly, unlike all other human viralvectors, there is no pre-existing immune response against baculovirus inhumans.

The construction of recombinant baculovirus is well documented.EP0340359, which is incorporated by reference, discloses a method ofobtaining a recombinant baculovirus incorporating a foreign gene throughuse of a transfer vector. The novel transfer vector incorporates arestriction site a short distance downstream of the N-terminus of thepolyhedrin gene body, into which a foreign gene may be cloned. Thenatural ATG start codon for the polyhedrin gene is not provided, suchthat the N-terminal polyhedrin coding sequence prior to the restrictionsite is retained but not capable of translation. A recombinantbaculovirus incorporating a foreign gene is derived from the transfervector by co-transfecting insect cells susceptible to baculovirusinfection with wild type baculovirus and the transfer vector.

Similarly U.S. Pat. No. 6,126,944, which is incorporated by reference,relates to the construction of baculovirus transfer vectors forefficient expression of foreign genes, and more particularly expressionof glycoprotein gG1 and gG2 of the Herpes Simplex virus. The foreigngene to be expressed is juxtaposed with the baculovirus polyhedrin geneat the translation initiation site, without the addition of furthernucleotides to the initiation site.

U.S. Pat. No. 5,750,383, which is incorporated by reference, discloses abaculovirus cloning system. The system is a marker rescue system usingan essential gene. The selected essential gene is inactivated. Cloninginto the baculovirus containing the null mutation is then achieved byusing the virus to infect wild type host cells that are co-infected witha plasmid containing a functional copy of the gene linked to a foreigngene under the control of a regulatable promoter. The baculovirus nullmutation is “rescued” by the rescue gene linked to a foreign gene. Thefunction of the essential gene is restored and the foreign gene isexpressed. An example of an essential gene is gp64 efp (envelope fusionprotein), that encodes a protein essential for viral infectivity andpropagation.

Although disclosing methods by which baculovirus may be manipulated theprior art is not related to the use of baculovirus as a viral vector fortargeted gene therapy.

We have developed a recombinant baculovirus which includes targetingsequences incorporated into the baculovirus genome which facilitate thedelivery of the baculovirus and thereby the therapeutic agent to aspecific cell type.

An example of a candidate gene for targeting is the baculovirus gp64, anextensively processed type 1 integral membrane glycoprotein. The role ofgp64 in baculovirus infectivity has been demonstrated by theneutralization of infectivity with antibodies specific to gp64. It hasalso been shown that gp64 is both necessary and sufficient for low pHactivated membrane fusion activity. Although conclusive data has beenlacking, indirect data on the role of gp64 in the infection cyclestrongly suggests that the protein is essential for infectivity of thebaculovirus.

The baculovirus gp64 envelope protein has been found to be sufficientlymutable to allow rapid insertion of new and more specific attachmentsequences, without perturbing its function as a carrier of geneticinformation. The gp64 envelope protein has a long loop, which haspreviously been used for antigen presentation and is ideal forinsertions. When modified gp64 is expressed together with wild type gp64in a mosaic membrane viral infectivity is not grossly affected.

According to a first aspect of the invention there is provided abaculovirus wherein the baculovirus genome has been modified to includea nucleic acid molecule which encodes a therapeutic agent and a nucleicacid molecule which encodes a polypeptide which functions to target saidbaculovirus to at least one cell type.

In a preferred embodiment said baculovirus genome is adapted foreukaryotic gene expression of said nucleic acid molecules.

Typically said adaptation includes, by example and not by way oflimitation, the provision of transcription control sequences (promotersequences) which mediate cell/tissue specific expression. These promotersequences may be cell/tissue specific, inducible or constitutive.

Promoter is an art recognised term and, for the sake of clarity,includes the following features which are provided by example only, andnot by way of limitation. Enhancer elements are cis acting nucleic acidsequences often found 5′ to the transcription initiation site of a gene(enhancers can also be found 3′ to a gene sequence or even located inintronic sequences and is therefore position independent). Enhancersfunction to increase the rate of transcription of the gene to which theenhancer is linked. Enhancer activity is responsive to trans actingtranscription factors (polypeptides) which have been shown to bindspecifically to enhancer elements. The binding/activity of transcriptionfactors (please see Eukaryotic Transcription Factors, by David SLatchman, Academic Press Ltd, San Diego) is responsive to a number ofenvironmental cues.

Promoter elements also include so called TATA box and RNA polymeraseinitiation selection (RIS) sequences which function to select a site oftranscription initiation. These sequences also bind polypeptides whichfunction, inter alia, to facilitate transcription initiation selectionby RNA polymerase.

Adaptations also include the provision of selectable markers andautonomous replication sequences which both facilitate the maintenanceof said vector in either the eukaryotic cell or prokaryotic host.

Adaptations which facilitate the expression of baculovirus encoded genesinclude the provision of transcription termination/polyadenylationsequences. This also includes the provision of internal ribosome entrysites (IRES) which function to maximise expression of baculovirusencoded genes arranged in bicistronic or multi-cistronic expressioncassettes.

These adaptations are well known in the art. There is a significantamount of published literature with respect to expression vectorconstruction and recombinant DNA techniques in general. Please see,Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory, Cold Spring Harbour, N.Y. and referencestherein; Marston, F (1987) DNA Cloning Techniques: A Practical ApproachVol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

In a preferred embodiment of the invention said eukaryotic expression isthrough the provision of cancer cell specific promoter elements.Preferably, said promoters are active in prostate cancer cells.

More preferably the promoter elements are selected from the group asrepresented in Table 1.

In a preferred embodiment of the invention said therapeutic agent is apolypeptide.

Preferably said polypeptide is a tumour suppressor polypeptide selectedfrom the following group represented in Table 2.

In a further preferred embodiment of the invention said polypeptide isan antigenic polypeptide.

Preferably a tumour rejection antigen precursor selected from thefollowing families represented in Table 3.

In a further preferred embodiment said polypeptide is a prostate tumourrejection antigen.

In a further preferred embodiment of the invention said polypeptide is acytotoxic polypeptide. For example pseudomonas exotoxin, ricin toxin,diptheria toxin (Genbank acc.#: A04646).

In a yet further preferred embodiment of the invention said polypeptideis a polypeptide which induces cell-cycle arrest.

Preferably said cell-cycle arrest polypeptide is selected from the grouprepresented in Table 4.

In a further preferred embodiment of the invention said therapeuticpolypeptide is a pharmaceutically active polypeptide. Preferably saidpolypeptide is a cytokine.

Preferably said cytokine is selected from the group represented in Table5.

In a yet further preferred embodiment of the invention said polypeptideis an antibody, or active binding fragment thereof, for example a Fabfragment.

Antibody fragments smaller than Fab fragments which bind cellulartargets are also within the scope of the invention. For example, singlechain Fv molecules (scFv).

These are engineered antibody fragments composed of a variable region ofthe heavy chain and a variable region of the light chain which arecoupled via a linker sequence, see Adams and Schier (1999) Journal ofImmunological Methods 249-260.

In a yet still further preferred embodiment of the invention saidpolypeptide is a polypeptide which induces apoptosis.

Preferably said apoptosis inducing polypeptide is represented in Table6.

In a yet still further preferred embodiment of the invention saidpolypeptide is a pro-drug activating polypeptide.

Preferably said prodrug activating polypeptide is represented in Table7.

In a still further preferred embodiment of the invention saidpolypeptide has anti-angiogenic activity. For example angiostatin, Tie2(Genbank acc. no: AF451865), endostatin (Genbank acc.no: NM130445).

In a further preferred embodiment of the invention said therapeuticagent is an antisense nucleic acid molecule.

As used herein, the term “antisense nucleic acid molecule” or“antisense” describes a nucleic acid which hybridizes underphysiological conditions to DNA comprising a particular gene or to anmRNA transcript of that gene and thereby, inhibits the transcription ofthat gene and/or the translation of that mRNA. The antisense moleculesare designed so as to interfere with transcription or translation of atarget gene upon hybridization with the target gene. Those skilled inthe art will recognize that the exact length of the antisense nucleicacid and its degree of complementarity with its target will depend uponthe specific target selected, including the sequence of the target andthe particular bases, which comprise that sequence.

It is preferred that the antisense nucleic acid be constructed andarranged so as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions.

Although nucleic acids may be chosen which are antisense to any regionof the gene or mRNA transcripts, in preferred embodiments the antisensenucleic acid correspond to N-terminal or 5′ upstream sites such astranslation initiation, transcription initiation or promoter sites. Inaddition, 3′-untranslated regions may be targeted. The 3′-untranslatedregions are known to contain cis acting sequences which act as bindingsites for proteins involved in stabilising mRNA molecules. These cisacting sites often form hair-loop structures which function to bind saidstabilising proteins. A well known example of this form of stabilityregulation is shown by histone mRNA's, the abundance of which iscontrolled, at least partially, post-transcriptionally.

The present invention, thus, contemplates a baculovirus genome which hasbeen modified by incorporation of an antisense nucleic acid to aspecific target sequence, for example a target sequence encoding acell-cycle regulatory gene, (eg p21 (Genbank acc.#: NM_(—)078467, c-myc(Genbank acc.#: D10493 and D90467), cyclin dependent kinase inhibitors,p16 (Genbank acc.#:NM058196), p15 (Genbank acc.#: BC002010), p18 (mousessequence Genbank acc.#: BC027026), or p19 (Genbank acc.#: NM_(—)079421)and apoptosis inhibitors such as caveolin.

In a further preferred embodiment of the invention said therapeuticagent is a double stranded RNA molecule. In this embodiment thebaculovirus genome would include a nucleic acid molecule under thecontrol of a first promoter positioned upstream (ie 5′ of the nucleicacid molecule) and a second promoter positioned downstream (ie 3′ of thenucleic acid molecule). The orientation of the promoters being such thatboth sense and antisense nucleic acid molecules are produced.

A technique to specifically ablate gene function is through theintroduction of double stranded RNA, also referred to as inhibitory RNA(RNAi), into a cell which results in the destruction of mRNAcomplementary to the sequence included in the RNAi molecule. The RNAimolecule comprises two complementary strands of RNA (a sense strand andan antisense strand) annealed to each other to form a double strandedRNA molecule. The RNAi molecule is typically derived from exonic orcoding sequence of the gene which is to be ablated. Alternatively saidRNAi molecule is derived from intronic sequences or the 5′ and/or 3′non-coding sequences which flank coding/exon sequences of genes. Recentstudies suggest that RNAi molecules ranging from 100-1000 bp derivedfrom coding sequence are effective inhibitors of gene expression.Surprisingly, only a few molecules of RNAi are required to block geneexpression which implies the mechanism is catalytic. The site of actionappears to be nuclear as little if any RNAi is detectable in thecytoplasm of cells indicating that RNAi exerts its effect during mRNAsynthesis or processing.

The exact mechanism of RNAi action is unknown although there aretheories to explain this phenomenon. For example, all organisms haveevolved protective mechanisms to limit the effects of exogenous geneexpression. For example, a virus often causes deleterious effects on theorganism it infects. Viral gene expression and/or replication thereforeneeds to be repressed. In addition, the rapid development of genetictransformation and the provision of transgenic plants and animals hasled to the realisation that transgenes are also recognised as foreignnucleic acid and subjected to phenomena variously called quelling(Singer and Selker, Curr Top Microbiol Immunol. 1995;197:165-77), genesilencing (Matzkeand Matzke, Novartis Found Symp. 1998;214:168-80;discussion 181-6. Review) and co-suppression (Stam et. al., Plant J.2000;21(1):27-42.

In a still further preferred embodiment said therapeutic agent is aribozyme.

A ribozyme is a catalytic RNA which is well known in the art. A ribozymecomprises a catalytic core having flanking sequences adjacent to thesequence which hybridises to the substrate RNA. The simplest catalyticcore is an RNA motif known as a hammerhead. Since the discovery ofcatalytic RNA there has been a desire to design ribozymes which have atargetted gene function such that disease gene mRNA's can be selectivelyablated.

In yet a further preferred embodiment of the invention the baculovirusgenome includes a nucleic acid molecule which encodes a polypeptidewhich binds the baculovirus to the cell surface of at least one celltype.

In a preferred embodiment of the invention said nucleic acid encodes apolypeptide selected from the following group: GnRH (Genbank acc.no:L03380), fibroblast growth factors; insulin and insulin-like growthfactors; neurotensin platelet derived growth factor (Genbank acc.no:NM_(—)002609 & NM_(—)006206); somatostatin (Genbank acc.no:BC032625).

In a preferred embodiment of the invention the nucleic acid encodingsaid polypeptide is inserted into the baculovirus genome at a site whichfuses said polypeptide to a baculovirus capsid polypeptide. Preferablythe capsid polypeptide is gp64.

Advantageously the fusion of the targeting polypeptide to a capsidpolypeptide will result in its presentation at the baculovirus particlesurface thereby presenting the baculovirus to said cell type and therebyfacilitating cell targeting.

According to a further aspect of the invention there is provided apharmaceutical composition comprising the baculovirus according to anyprevious aspect or embodiment of the invention. Preferably saidcomposition is for use in the manufacture of a medicament for thetreatment of cancer, ideally prostate cancer.

According to a yet further aspect of the invention there is provided amethod of treatment comprising the administration of a therapeuticallyeffective amount of the baculovirus according to the invention.

In a preferred method of the invention said treatment is cancer,preferably prostate cancer.

An embodiment of this invention will now be provided by example only andwith reference to the following materials, methods, vectors and figures:

FIG. 1 illustrates reporter gene expression by baculoviral based vectorAcMNPV;

FIG. 2 illustrates baculoviral expression of AcMNPV in insect cells,Sf9;

FIG. 3 illustrates the lack of expression of baculoviral encoded genesin mammalian cells;

FIG. 4 is baculovirus vector pBAsurf-1 MCS2;

FIG. 5 is baculovirus vector pBAsurf-1 GnRH(MKII); and

FIG. 6 is baculovirus vector pBacMam2 EGFP.

MATERIALS AND METHODS

Targeting baculoviruses are generated in two stages (i) by generation ofa transfer vector in a bacterial plasmid, which is multiplied inbacteria, and whose DNA sequence in determined to verify the insertionof the recombinant DNA sequence; and (ii) recombination of the transfervector, via homologous non essential region on either side of the gp64recombinant, into a multiply cut Bv genome by cotransfection intorecipient insect cells (sf9 or sf21).

An example of the experimental procedure is as follows.

The DNA sequence encoding the minimal peptide required for receptorbinding for the GnRH and neurotensin receptors was determined and a DNAoligonucleotides for both strands were chemically synthesised, includingPstI and KpnI restriction endonuclease sites to facilitate insertioninto the pBACsurf vector (FIG. 4). The synthesised oligonucleotides werethen ligated into the pBACsurf vector via these restriction endonucleasesites The sequences of the peptides and a map of the vector are shownbelow, see FIG. 5 and FIG. 6: GnRH peptide coding sequenceCTGCAGCAACATTGGAGCTACGGCTTGCGCCCGGGCGCGGTACC GnRH amino acid sequenceLeuGlnGlnHisTrpSerTyrGlyLeuArgProGlyAlaVal Neurotensin peptide codingsequence CTGCAGGAATTGTACGAAAACAAACCGCGCCGCCCGTACATTTTGGCGGT ACCNeurotensin peptide LeuGlnGluLeuTyrGluAsnLysProArgArgProTyrIleLeuAla Val

Full DNA sequence data for the constructs should be available for thefinal constructs, particularly the segment of the gp64 fusion protein.

The sequenced plasmid is then recombined into the Bacvector-1000 triplecut baculovirus DNA (Novagen) by cotransfection into sf21 cells. Theresulting baculoviruses are only viable if recombination has occurred,and are diploid for the gp64 gene, as insertion does not occur in thenative gp64 locus. This is essential to preserve high infectivity of thebaculovirus, and has been observed in other systems eg HIV, where envprotein modification can be carried out.

A further modification of the pBACsurf vector was carried out, in orderto facilitate a single recombination step for both of the humanisingsequences (ie human promoter and cell surface attachment), whereby asecond multiple cloning site (MCS2) was inserted into the recombinationarea, which contains unique (ie single cut for the plasmid) RE sites.This is shown below:

The alternative method of deriving the multiple recombinants is toco-transfect the promoter vector pBACMAM2 with the singly modifiedpBACsurf with the Bacvector 1000 triple cut DNA into sf21 insect cells,and to screen for double recombinant viruses by polymerase chainreaction. This is the method of choice when large (>3 kb) promoterfragments are inserted, as the capacity of the pBACsurf (MCS2) vector islimited. Viral DNA from the recombinant plaques therefore ischaracterised by a wild-type PCR product and a larger product from theinsertion recombinant. The sense of the insertion is verified by directDNA sequencing of the purified PCR product.

Promoter fragments are inserted into the pBACMAM vector to replace thehybrid CAG promoter (CMV enhancer (within Genbank acc.#: AF477200)),Chicken beta actin promoter (Genbank acc.#: E02199) and rabbit betaglobin terminator (Genbank acc.#:AX451706). To facilitate this a generalinsertion construct was prepared in pT7 blue vector, such that thepromoter is inserted upstream of either indicator genes (for activity inhuman cells such as the enhanced green fluorescent protein (EGFP)(Genbank acc.#: U57609) or a hybrid consisting of the EGFP fused to thecommon bacterial indicator chloramphenicol acetyl transferase or CATgene (Genbank acc.#: D14641). This construct is then excised from thepT7 blue carrier and inserted via SphI/SwaI and HindIII/BcII sites intothe pBACMAM vector. The use of the multiple cloning site in the pBACMAMvector (thus retaining the BgIII, StuI, Sae8387, NotI, KpnI, SmaI, Bsu36and MacI sites) and inserting the promoter construct upstream of theRabbit beta globin terminator (Genbank acc.#: AX451706) is alsopossible.

Gene maps are created in Gene Construction Kit v2 (Textco Inc, USA)

EXAMPLE 1

The baculovirus used to infect one insect and two human cell lines(Table 2) was a recombinant AcMNPV engineered to carry the EGFP reportergene under the control of the strong mammalian CAG promoter. The humancells were inspected 48 Hrs post infection, using fluorescentmicroscopy, to visualise the EGFP expression (FIG. 1). Significantly,visual inspection suggests the infection efficiency in vitro for theLNCaP prostate cancer cells was equivalent to that of the 293 cells (25%by flow cytometry analysis, data not shown)—no appreciable EGFPexpression can be seen from the CAG promoter in insect Sf-9 cells (datanot shown). Total RNA was extracted from all cells and RT-PCR carriedout using the six sets of primers designed against baculoviral mRNA(Table 1), plus a G3PDH primer set as a positive control for the humancells.

In the Sf9 cells, expression of four out of the six genes examined wasdetected via RT-PCR in infected cells and but not in uninfected cells(FIG. 2) indicating baculoviral specific expression. The exceptions tothis were the ubiquitin gene and the egt gene. Ubiquitin expression wasdetected in both infected and uninfected insect cells using the primersdesigned against baculovirus ubiquitin (FIG. 2), although the levels inuninfected cells were much lower. This can be explained in thatubiquitin is a highly conserved molecule and there may be enoughhomology between insect and baculoviral ubiquitin to produce an RT-PCRproduct of the same size. It is also possible that the baculoviralubiquitin may actually have derived from its insect cell host duringevolution. The other exception was the egt gene which was not expressedin either infected or uninfected cells (FIG. 2). Egt is expressed veryearly in the baculoviral life cycle to prevent larval moulting and Sf9cells are derived from pupal ovarian tissue. Therefore, it can behypothesised that there are factors in the larval form of the host whichare not found in the pupal stage and are necessary for egt expression.

EXAMPLE 2

For expression analysis in human cells, the 293 human embryonic kidneycell line was chosen due to its reported ease of infection withbaculovirus (Condreay J P, Proc.Natl.Acad.Sci. USA 1999; 96: 127-132;Boyce F M, Bucher N L R. Proc.Natl.Acad.Sci. USA 1996; 93:2348-2352) andthe wide range of gene it can be induced to express. Additionally, theLNCaP prostate cancer cell line (derived from lymph node metastases) waschosen as a model for prostate cancer gene therapy—especially as thesecells are difficult to transduce by liposome based techniques. Theresults for the RT-PCR for both of these cells lines show that none ofthe six baculoviral genes examined are expressed in either uninfected orinfected cells in either cell line (FIG. 3), despite the high levels ofexpression of the CAG controlled EGFP. Messenger RNA for thehousekeeping gene, G3PDH, was also found in uninfected and infectedcells in both of the cells lines using significantly fewer PCR cycles(25 cycles compared to 35 when screening for the baculoviral mRNA).These results indicate that a representative portion of the endogenousgenes found in baculovirus, most of which are highly expressed in thenormal eukaryotic baculovirus host environment and can interact withessential human cell processes, are not expressed when the virus is usedto infect human cells. In contrast protein can be effectivelysynthesised from a reporter gene under the control of a mammalianpromoter. This lack of baculoviral gene expression in human cellsfurther underlines the safety of the baculovirus as a vector for genetherapy, particularly for transient suicide gene protocols, sinceneither baculoviral gene expression or genome integration will be alikely complication. TABLE 1 Promoter sequence DNA Accession numberProstate androgen BC026274 or NM005551 regulated transcript 1 Prostatetransglutaminase, BC007003 Prostase XM031805 Prostate-derived Ets factorAF071538 Prostatic acid phosphatase X53605 Pr LeuZip PAGE-4 AF275258 DD3NKX3.1 AF247704 probasin AX259949 prostate-specific antigen AJ459782prostate-specific XM165392 membrane antigen prostate stem cell antigenXM030742 prostate carcinoma tumor NM006499 antigen-1 AIPC AF338650Trp-p8 AC005538 E2F4 AF527540 Daxx AF015956 TRPM-2 NM001831 PART-1nm016590 TMPRSS2 Bomesin Steap Nm 012449 TARP Af151103 PcGEM1 Af223389

TABLE 2 Tumor DNA accession suppressor Polypeptide number p53 AF136270Retinoblastoma APC polypeptide NM000038 DPC-4 polypeptide U73825 BRCA-1polypeptide BRCA-2 polypeptide WT-1 polypeptide XM_034418 MMAC-1polypeptide XM083839 Familial polyposis coli NM000038 polypeptide

TABLE 3 Tumor Rejection DNA Accession Antigen Precursor Family numberMAGE XM066465 BAGE NM001187 GAGE NM_003785 DAGE Q99958

TABLE 4 Cell-CycleArrest DNA accession Polypeptide number p21 NM078467p16 NM058196 p15 BC002010 p18 BC027026 p19 NM079421 PTEN AF143312

TABLE 5 Cytokine DNA Accession number growth hormone leptinerythropoietin prolactin IL-2 XM_035511 IL-3 U81493 IL-4 AF395008 IL-5AF353265 IL-6 AF039224 IL-7 NM000880 IL-9 AF361105 IL-10 BC022315 IL-11BC012506 the p35 subunit of IL-12 AF101062 IL-13 AF377331 IL-15 AF031167G-CSF E09569 GM-CSF M13207 CNTF E09734 CT-1 XM096076 LIF XM009915oncostatin M NM020530 IFNα J00207

TABLE 6 Apoptosis DNA Accession inducing polypeptide number P53 AF136270adenovirus E3.11.6K adenovirus E4 adenovirus f4 caspase Fas ligandE11157 C-Cam 1 XM113980 ODC NM052998 OAZ XM037830 spermidine/spermineN1- BC002503 acetyltransferase ZNF145 NM006006 PTEN phosphatase AF143312androgen receptor NM_000044 Bcl2 family members.

TABLE 7 Prodrug DNA Accession Activating polypeptide number cytosinedeaminase AL627278 thymidine kinase AB078742 nitroreductase RdxAAY063488 Cytochrome P450 NM_000761 CYP1A2 CYP2E1 AB052259 CYP3A4AF209389

TABLE 1 Baculoviral genes chosen for expression analysis Stage of LifeSize of PCR Cycle Human Primer Product Baculoviral Gene Gene FunctionExpressed Homologue Name (bp) Proliferating cell nuclear Stimulates DNAreplication and late Early Yes pcna 308 antigen gene expression DNApolymerase DNA replication Early Yes DNA-pol 247 Ubiquitin Blocksubiquitin dependent proteolysis Late Yes ubi 142 gp37 (p34.8) Spindlebody protein Late/Very Late No gp37 347 p10 Viral lysis (?) Very Late Nop10 202 Ecdysteroid UDP- Blocks larval moulting Early No egt 281glucosyltransferase

TABLE 2 Cell lines used to investigate native baculovirus geneexpression. Cell Line Description Sf-9 Insect cell line, derived frompupal ovarian tissue of the fall army worm, Spodoptera frugiperda 293Human cell line derived from human embryonic kidney and transformed toimmortality by adenovirus 5 LNCaP Androgen-dependent, non-metastatic,non-tumourigenic human prostate cancer cell line derived from lymph nodemetastasis

1. A baculovirus wherein the baculovirus genome has been modified tocomprise a first polynucleotide which encodes a therapeutic agent and asecond polynucleotide which encodes a polypeptide which functions totarget said baculovirus to at least one cell type.
 2. A baculovirusaccording to claim 1 wherein said genome is adapted for eukaryotic geneexpression of said first and second polynucleotides.
 3. A baculovirusaccording to claim 2 wherein the expression of said first polynucleotideis controlled by a cancer specific promoter.
 4. A baculovirus accordingto claim 3 wherein said cancer specific promoter is a prostate cancercell specific promoter.
 5. A baculovirus according to claim 3 whereinsaid promoter is a promoter listed in Table
 1. 6. A baculovirusaccording to claim 1 wherein said therapeutic agent is a polypeptide. 7.A baculovirus according to claim 6 wherein said therapeutic agent is atumor suppressor polypeptide listed in Table
 2. 8. A baculovirusaccording to claim 7 wherein said therapeutic agent is an antigenicpolypeptide.
 9. A baculovirus according to claim 8 wherein saidantigenic polypeptide is a prostate tumor rejection antigen.
 10. Abaculovirus according to claim 6 wherein said therapeutic agent is acytotoxic polypeptide.
 11. A baculovirus according to claim 10 whereinsaid cytotoxic polypeptide is selected from the group consisting of:pseudomonas exotoxin; ricin toxin; and diptheria toxin (Genbank ace.#:A04646).
 12. A baculovirus according to claim 6 wherein said therapeuticagent is a polypeptide which induces cell-cycle arrest.
 13. Abaculovirus according to claim 12 wherein said therapeutic agent is apolypeptide listed in Table
 4. 14. A baculovirus according to claim 6wherein said therapeutic agent is a pharmaceutically active polypeptide.15. A baculovirus according to claim 14 wherein said therapeutic agentis a cytokine.
 16. A baculovirus according to claim 15 wherein saidtherapeutic agent is a cytokine listed in Table
 5. 17. A baculovirusaccording to claim 6 wherein said therapeutic agent is an antibody oractive binding fragment thereof.
 18. A baculovirus according to claim 17wherein said fragment is a Fab fragment.
 19. A baculovirus according toclaim 6 wherein said therapeutic agent is a polypeptide which inducesapoptosis.
 20. A baculovirus according to claim 19 wherein saidtherapeutic agent is an apoptosis-inducing polypeptide listed in Table6.
 21. A baculovirus according to claim 6 wherein said therapeutic agentis a pro-drug activating polypeptide.
 22. A baculovirus according toclaim 21 wherein said therapeutic agent is a prodrug-activatingpolypeptide listed in Table
 7. 23. A baculovirus according to claim 22wherein said prodrug-activating polypeptide has antiangiogenic activity.24. A baculovirus according to claim 23 wherein said prodrug-activatingpolypeptide is selected from the group consisting of angiostatin(Genbank accANM007037), Tie2 (Genbank acc.#: AF451865), and endostatin(Genbank acc.#: NM130445).
 25. A baculovirus according to claim 1wherein said therapeutic agent is an antisense nucleic acid molecule.26. A baculovirus according to claim 25 wherein said antisense nucleicacid molecule binds a nucleic acid molecule encoding a cell-cycleregulatory gene.
 27. A baculovirus according to claim 26 wherein saidantisense nucleic acid molecule binds a cell-cycle regulatory genelisted in Table
 4. 28. A baculovirus according to claim 25 wherein saidantisense nucleic acid molecule binds a nucleic acid molecule encodingan apoptosis inhibitor.
 29. A baculovirus according to claim 28 whereinsaid apoptosis inhibitor is caveolin (Genbank ace.#: AF095591)
 30. Abaculovirus according to claim 1 wherein said therapeutic agent is adouble stranded RNA molecule.
 31. A baculovirus according to any claim 1wherein said therapeutic agent is a ribozyme.
 32. A baculovirusaccording to any of claim 1 wherein said baculovirus genome furthercomprises a third polynucleotide which encodes a polypeptide which bindsthe baculovirus to the cell surface of at least one cell type.
 33. Abaculovirus according to claim 32 wherein said nucleic acid encodes apolypeptide selected from the group consisting of. GnRH (Genbank ace.#:L03380), fibroblast growth factors; insulin, an insulin-like growthfactor; neurotensin; platelet derived growth factor (Genbank acc.#:NM002609 & NM 006206); and somatostatin (Genbank acc.#: BC032625).
 34. Abaculovirus according to claim 32 wherein said third polynucleotide isinserted into the baculovirus genome at a site such that saidpolypeptide which binds the baculovirus to the cell surface is fused toa baculovirus capsid polypeptide.
 35. A baculovirus according to claim34 wherein said capsid polypeptide is gp64.
 36. A pharmaceuticalcomposition comprising the baculovirus according to claim 1 and apharmaceutically acceptable excipient. 37-38. (Cancelled)
 39. A methodof treatment comprising the administration of a therapeuticallyeffective amount of a pharmaceutical composition of claim
 36. 40. Amethod according to claim 39 wherein said method is for the treatment ofcancer.
 41. A method according to claim 40 wherein said cancer isprostate cancer.